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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
2015-08-21 to 2015-10-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted 3 October 2008
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-(C8-10 acyl) derivs., hydroxides, inner salts
- Analytical purity: 34.65% active ingredient (betain)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 27-29 days
- Weight at study initiation: 92-100 g for males and 80-95 g for females at arrival
- Fasting period before study: no
- Housing: up to 5 of one sex to a cage, in clear polisulphone solid bottomed cages; nesting material was provided inside suitable bedding bag and changed at least twice a week
- Diet (e.g. ad libitum): powdered laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: approx. 3 wk

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 55±15
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: purified water (softened water by reverse osmosis)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was dissolved in the vehicle. The formulations were prepared daily at concentrations of 10, 30 and 50 mg/mL. Concentrations were calculated and expressed in terms of test item corrected against the declared purity.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw/d
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The proposed formulation procedure for the test item was checked in the range from 10 to 100 mg/mL by chemical analysis (concentration) and a 28 hour and 8 day stability at room temperature was verified in the same range, to confirm that the method was suitable and stability was satisfactory.
Final results for all levels were within the acceptability limits for concentration (90-110%).
Samples of the formulations prepared on Weeks 1 and 4 were analysed to check the concentration. Results of the analyses were within the acceptability
limits for concentration of solutions (90-110%)
Duration of treatment / exposure:
4 weeks + recovery period of 2 weeks
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 500 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
5 male and 5 female; control and high dose groups included 5 additional animals per sex to be sacrificed after 2 weeks of recovery
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results with similar substances
- Rationale for animal assignment (if not random): computerised stratified randomisation to give approximately equal initial group mean body weights
- Rationale for selecting satellite groups: to assess recovery from any treatment-related effects
- Post-exposure recovery period in satellite groups: 2 weeks
Positive control:
n.a.

Examinations

Observations and examinations performed and frequency:
MORTALITY: Yes
- twice daily

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily, approximately 1 hour after dosing

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before commencement of treatment and at least once per week from the start of treatment
- parameters: gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, unusual respiratory pattern)

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly starting from the day of allocation to treatment group and just prior to necropsy

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of Week 4 of treatment, prior to necropsy / at the end of Week 2 of the recovery period
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, White blood cell count, Differential leucocyte count (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells), Platelets, Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of Week 4 of treatment, prior to necropsy / at the end of Week 2 of the recovery period
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Bile acids, Phosphorus, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during Week 4 of treatment and once during Week 2 of recovery
- Dose groups that were examined: all groups
- Battery of functions tested: sensory activity / grip strength / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)
Other examinations:
The testes and epididymides of main group animals were cut at 2-3 µm thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed initially in all animals in the control and high dose groups dying during the treatment period or killed at the end of the 4 weeks of treatment.
Moreover, the histopathological evaluation of the oestrous cycle in the uterus/ cervix and vagina from all animals in the control and high dose groups of the
main phase was performed.
Statistics:
Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test.
If the data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied.
The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study.
No clinical signs were seen during the study.
No toxicological relevant changes were observed at the weekly detailed clinical signs.
Statistically significant decreases in rearing were seen in males receiving 300 and 500 mg/kg bw/day during Weeks 3 and 4 of treatment, increases were seen
in females receiving 100 and 300 mg/kg bw/day during Week 3 and in females receiving 500 mg/kg bw/day during Week 4 of treatment, when compared to
controls. Since the direction of changes was opposite in the two sexes, the above mentioned changes were considered incidental and not treatmentrelated.

BODY WEIGHT AND WEIGHT GAIN
No differences in body weight were seen between treated and control animals during the study.

FOOD CONSUMPTION
No differences in food consumption were seen between treated and control groups during the study.

HAEMATOLOGY
Dosing phase
Lymphocytosis and/or monocytosis were recorded in two females dosed at 300 mg/kg bw/day and 3 females receiving 500 mg/kg bw/day. When compared
with mean control data, individual changes were between 61% and 93% for lymphocytes and 137% and 150% for monocytes.
The statistically significant increase of eosinophils observed in females dosed at 300 mg/kg bw/day was not dose-related, therefore considered incidental.
Recovery phase
After the recovery period, one female showed lymphocytosis and neutrophilia. When compared with mean control data, the individual change was of 49% and 3.7 fold, respectively. Concerning the other treated animals, lymphocytes showed complete reversibility and monocytes data were comparable with controls, even though values were similar to those observed during the dosing phase.
The statistically significant differences recorded between control and treated animals (reticulocytes and lymphocytes relative count in males, mean corpuscular
haemoglobin in females) were not observed during the dosing phase, therefore considered unrelated to treatment.

No changes in coagulation parameter were seen in control and treated animals.

CLINICAL CHEMISTRY
Chloride and sodium showed a statistically significant decrease in females dosed at 300 and/or 500 mg/kg bw/day, when compared to controls. Due to the
minimal severity (up to 2%), these findings were considered of no toxicological relevance.
The statistically significant changes recorded in animals dosed at 100 and/or 300 mg/kg bw/day (bilirubin, bile acids and inorganic phosphorus in males; urea
in females) were not dose-related, therefore considered unrelated to treatment.

NEUROBEHAVIOUR
No relevant differenecs between treated and control groups were seen in landing foot splay.
Changes in grip strength were considered incidental and not treatment-related, since the direction of changes was opposite in the two sexes: decreases in
males and increases were seen in females, receiving 500 mg/kg bw/day, when compared to controls.
No differences between treated and control groups were evident at the motor activity measurements.

ORGAN WEIGHTS
No relevant differences were seen in terminal body weights of treated animals respect to controls.
No toxicological relevance was attributed to the statistically significant increase in absolute (13%) and/or relative (9-10%) kidneys weight seen in females treated at 300 and/or 500 mg/kg/day not to the statistically significant increase in relative (up to 7%) kidneys weight seen in males treated at 300 and 500 mg/kg/day, when compared to controls, since these increases were slight and no changes were detected at the histopathological evaluation.
No other differences were seen in treated groups when compared to controls.

GROSS PATHOLOGY
No relevant changes were noted in treated animals. The sporadic changes observed in few treated animals could be considered incidental.

HISTOPATHOLOGY: NON-NEOPLASTIC
No treatment-related changes were noted. All observed changes in organs and tissues are considered as incidental-age related findings, characteristically seen in untreated Sprague Dawley rats of the same age or comparable with control animals. Seminiferous tubules were evaluated with respect to their stage in
the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
Normal physiology of the oestrous cycle (oestrous, metestrous, diestrous and proestrous) was noted in control and treated females. The morphological changes seen were normal when compared to each “oestrous phase” in the ovaries, uterus/cervix and vagina.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: no treatment-related adverse effects up to and including the highest tested dose level

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
On the basis of the results obtained in this study, the dose level of 500 mg/kg bw/day was considered the NOAEL.
Executive summary:

In a subacute toxicity study according to OECD guideline 407 (2008) and EU method B.7 (2008) C8-10 Alkylamidopropyl betaine (34.65% a.i.) was administered to 5Hsd: Sprague Dawley SDrats/sex/dose in purified water by gavage at dose levels of 0 (control), 100, 300 and 500 mg/kg bw/day for 28 consecutive days. Control and high dose groups included 5 additional animals per sex to be sacrificed after 2 weeks of recovery.

No mortality occurred. No clinical signs and no changes were observed at the weekly detailed clinical observations. Neurotoxicity assessment did not reveal any treatment-related effects. No changes on body weight and food consumption were noted.

The lymphocytosis and monocytosis seen in single females dosed at 300 and/or 500 mg/kg bw/day showed reversibility at the end of the recovery period or comparability to control data. No toxicological relevant effects in coagulation and clinical chemistry parameters were observed. No differences were reported in terminal body weights and organ weights between treated and control animals and no treatment-related changes were noted at macroscopic and microscopic observations.

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

Normal physiology of the oestrous cycle (oestrous, metestrous, diestrous and proestrous) was noted in control and treated females. The morphological changes seen were normal when compared to each “oestrous phase” in the ovaries, uterus/cervix and vagina.

On the basis of the results obtained in this study, the dose level of 500 mg/kg bw/day was considered the NOAEL (No Observed Adverse Effect Level).